System for staining fungi and protozoa

ABSTRACT

The present invention provides a method for the staining of fungi and microsporidia for observation with a light microscope based upon the presence of chitin in the composition of these organisms. With the method of the present invention a sample to be analyzed is treated with a solution of Ponceau S and Stains-all dye. The sample is then selectively decolorized and rinsed. The resulting sample is examined with a light microscope, or photographed for a permanent record, to identify the presence of a variety of microorganisms, to include fungi and microsporidia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of prior application Ser. No.10/249,663, entitled “Method for Staining Fungi and Protozoa”, filed onApr. 29, 2003, the contents of which are incorporated herein byreference, which claims the benefit of U.S. Provisional PatentApplication 60/319,214, entitled, “Method for Staining Fungi andProtozoa”, filed Apr. 29, 2002.

BACKGROUND OF THE INVENTION

This invention is a novel method of staining and detecting protozoa andfungi for observation under a light microscope, and with application tothe qualitative and quantitative detection of microsporidia inenvironmental water samples.

Fungi and microsporidia can be differentiated from other microorganismsbased on their chitin content. Current dyes used to stain chitin infungi and microsporidia are based on fluorescent optical brighteners(Review by Ruchel et al., 2001), requiring a fluorescent microscope forobservation. Details are not observable by this method.

A need exists in the art for the identification of the characteristicstructures in microsporidian spores, including but not limited to thickspore walls, vacuole, belt-like stripe and sporoplasm, that can beimplemented using a light microscope, and recorded using a digitalcamera.

SUMMARY OF INVENTION

The present invention provides a method for determining the presence orabsence of a microorganism in a sample suspected of containing amicroorganism. In a preferred embodiment, a sample is provided to beanalyzed, the sample is treated with a protein stain, and the sample isthen treated with a cationic carbocyanine dye and examined formicroorganisms.

In an additional embodiment, the protein stain used to treat the sampleis Ponceau S, and preferably is a solution of 0.1% Ponceau S in 5%acetic acid in water.

In an additional embodiment, the cationic carbocyanine dye used to treatthe sample is Stains-all, and preferably is a solution of Stains-all inmethanol diluted 1:10 in a solution of deionized water, acetic acid, andmethanol at 50:10:40.

In an additional embodiment of the present invention, an additional stepof selectively decolorizing the sample is included. Selectivelydecolorizing the sample is facilitated through the application of adecolorizing solution. The decolorizing solution can be a solution ofsodium dodecyl sulfate in phosphate buffered saline. Preferably thesodium dodecyl sulfate being 0.25% in phosphate buffered saline.Additionally, the decolorizing solution can be a solution of acidmethanol, in a preferred solution of 50:10:40 deionized water, aceticacid, and methanol.

In an additional preferred embodiment of the present invention, anadditional step of rinsing the sample prior to examining the sample inincluded. The rinsing step is facilitated by the application of asolution of acid methanol to the sample and subsequent treatment of thesample with a solution of phosphate buffered saline. Preferably thesolution of acid methanol employed in the rinsing step is 50:10:40deionized water, acetic acid, and methanol and the solution of phosphatebuffered saline contains about 0.05% Tween 20.

In a preferred method of the present invention for the determination ofthe absence or presence of a microorganism in a sample suspected ofcontaining a microorganism, a provided sample is treated with a solutionof Ponceau S and Stains-all dye solution. The sample is then selectivelydecolorized, rinsed and examined for microorganisms.

A kit for detecting the presence of a microorganism is provided by thepresent invention in which is provided a protein stain and a cationiccarbocyanine dye.

In an additional embodiment of the kit, the protein stain is Ponceau S,and preferably a solution of 0.1% Ponceau S in 5% acetic acid in water.

In yet another embodiment of the kit of the present invention, thecationic carbocyanine dye is Stains-all, and preferably a solution ofStains-all in methanol diluted 1:10 in a solution of deionized water,acetic acid, and methanol at 50:10:40.

In an additional embodiment, the kit of the present invention includes aselective decolorizing solution. The decolorizing solution being sodiumdodecyl sulfate in phosphate buffered saline, and preferably a solutionof 0.25% sodium dodecyl sulfate in phosphate buffered saline.Additionally, the decolorizing solution can be a solution of acidmethanol, preferably a solution of 50:10:40 deionized water, aceticacid, and methanol.

In yet another embodiment, the kit of the present invention includes arinsing solution. The rinsing solution being acid methanol, preferablythe solution of acid methanol is 50:10:40 deionized water, acetic acid,and methanol.

In another embodiment, the kit contains phosphate buffered salinesolution. Preferably, the phosphate buffered saline solution being 0.05%Tween 20.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and objects of the invention,reference should be made to the following detailed description, taken inconnection with the accompanying drawings, in which:

FIG. 1 are photographic slides of the staining of Penicillium notatumusing the present invention.

FIG. 2 are photographic slides of the staining of Aspergillus nigerusing the present invention.

FIG. 3 are photographic slides of the staining of Escherichia coli,streptocoocus mutans, and microsporidia using the present invention.

FIG. 4 are photographic slides of the staining of Cryptosporidium usingthe present invention.

FIG. 5 are photographic slides of the staining of Microsporidia usingthe present invention.

FIG. 6 are photographic slides of the staining of Rhizopus stoloniferusing the present invention.

FIG. 7 are photographic slides of background destaining using thepresent invention.

FIG. 8 is a flow diagram demonstrating a method identified as Protocol 1of the present invention.

FIG. 9 is a flow diagram demonstrating a method identified as Protocol 2of the present invention.

FIG. 10 is a flow diagram demonstrating a method identified as Protocol3 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The method of the present invention is based on three originalobservations made, including: (1) the strong binding of Ponceau S tochitosan, a derivative of chitin, and presumably otherglucoaminoglycans; (2) the stable binding of Stains-all to Ponceau S;(3) the selective removal of dyes from non-chitin containingmicroorganisms by a PBS solution containing sodium dodecyl sulfate(SDS). Based on this method, convenient staining kits for fungi andmicrosporidia can be prepared and commercialized for use in research andclinical laboratories, in water analysis, and in food quality control.This method is also amenable to the staining of tissue sections, andautomation for the routine analysis of a large number of environmentalwater samples.

New properties for the dye Stains-all have been observed in thelaboratory setting. The dye complexes readily with the dye Ponceau S,causing a shift in the absorption spectrum of both the Ponceau S and theStains-all dye. Additionally, new properties for the dye Ponceau S havebeen discovered. This dye is widely used for the staining of proteins invarious applications, but it has been heretofore unknown in the priorart that this dye can stain strongly and permanently chitosan, aderivative of chitin. Subsequent to this finding, a protocol wasinvented to put these properties to use in the staining of fungi, andmicrosporidia as these organisms are known to contain chitin. Using thisprotocol, these organisms can be stained and identified, even in acomplex mixture.

The preferred protocol of the present invention comprises the followingsteps (1) preparation of tissue section on slide, or application of thesamples to be analyzed onto microscopic slide, followed by drying; (2)sequential addition the dye Ponceau S and the dye Stains-all, followedby drying (3) selective decolorization by addition of SDS to destainnon-chitin containing cells or microorganisms followed by drying; (4)sequential rinsing of the slide by acid-methanol, methanol, PBScontaining Tween 20, then methanol. The drying step may be performed ona heating block (50° C.) to speed up the process. A light microscope isused for observation.

The staining method of the present invention uses Ponceau S and theStains-all dyes. Ponceau S is also known as C.I. Acid Red 112, PonceauRed and Ponceau X Extra. The chemical formula for Ponceau S is3-hydroxy-4((2-sulfo-4-((−4-sulfophenyl(azo)phenyl-)azo)2,7naphthalenedisulfonic acid tetrasodium salt. The dye Stains-all, acationic carbocyanine dye, [7423-31-6]1-Ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphto[1,2-d]thiazoliumbromide, is a sensitive stain for various biochemical componentsincluding nucleic acid, hyaluronic acid phosphoproteins andnon-phosphorylated proteins, and acid mucopolysaccharides.

Different colors are obtained depending on the nature of the biochemicalcomponent. The Stains-all dye interacts with these compounds formingdifferent complexes absorbing at various wavelengths (Kay et al., 1964;Green et al., 1973). Application of the Stains-all dye is limited to theelectrophoretic characterization of nucleic acids, proteins(phosphorylated and non-phosphorylated), and substituted polysaccharidesin polyacrylamide or composite agarose and polyacrylamide gels (Green etal., 1973; Dalberg et al., 1969; Bader et al., 1972). Stains-all can beused to simultaneously stain nucleic acids, proteins, conjugatedproteins and polar lipids (Green, 1975). Although, Stains-all is asensitive and potentially useful as a differential dye, it has not beenused to stain microorganisms. One disadvantage associated with the dyeis that it fades rapidly.

Thus, the dye Stains-all used in combination with Ponceau S to stainmicrosporidia and fungi produces new and unexpected results. By stainingwith Ponceau S alone, it is not possible to identify these organismsbecause Ponceau S stains both proteins and polysaccharides in pink orred. As stated above, Stains-all can differentially stain a number ofmacromolecules, but has not yet been used to stain microorganisms. Thisdye is also unpopular due to its propensity to fade away. According tothe invented protocol, Ponceau S is used first to stain proteins andmore strongly chitin before the addition of Stains-all. The latter dyecomplexes in situ with Ponceau S, enhancing the coloration and providingdifferent tints due to its binding to other components. The resultingeffect is a stable coloration, withstanding the selective decolorizationstep, and allowing the detection and analysis of fungi andmicrosporidia, even in a complex mixture of microorganisms.

Protocol #1

Protocol #1 as shown in FIG. 8, the preferred staining method as taughtby the present invention comprises five solutions: (1) solution of 0.1%Ponceau S in 5% Acetic acid (Ponceau S, Sodium salt is a product ofSigma Chemical Company, St. Louis, Mo.); (2) solution comprising amixture (1:9, v:v) of 0.2% Stains-all (Stains-all is a product of AcrosOrganics, NJ) in Methanol (stock solution), which is diluted 1:10 in asolution of acid-methanol (solution 4) before use; (3) solutioncomprising 0.25% SDS in PBS; (4) solution of Deionized water:AceticAcid: Methanol (50:10:40); (5) solution of PBS containing 0.05% Tween20. Preferably, the solutions are supplied in drop-dispensing bottles tosimplify the procedure. Additional requirements include Methanol, aheating block set at 50° C., a light microscope, and a digital camera totake micrographs of the slides (Olympus C3030Z or higher).

In an exemplary method, protocol # 1, of the present invention,comprises:

APPLICATION OF THE SAMPLE: apply tissue section or aqueous samplecontaining microorganisms (ex. suspension in PBS) is applied onto amicroscopic slide 10, and allowed to dry 15 at room temperature or on aheating block (50° C.);

STAINING: one or more drops of solution 1 20 is added to cover thesample for 10 sec, followed by 2 or more drops of solution 2 25 to coverthe sample for 10 sec, pouring off the excess solution and drying 30 atroom temperature or on a heating block;

SELECTIVE DECOLORIZATION: 2 or more drops of solution 3 are added tocover the sample 35, and left to stand for approximately three minutesuntil decolorization of extraneous materials. Then the slide ispermitted to air-dry 45 at room temperature or on a heating block (50°C.). Alternatively, an equal volume of acid methanol (solution 4) isadded 40 and left in contact for 10 sec, followed by pouring off theexcess and rinsing with methanol; Rinsing: The slide is sequentiallyrinsed 50 with solution 4 added dropwise to cover the sample for 10 sec,followed by methanol, then with solution 5 added dropwise to cover thesample for 10 sec, followed by three rinses with methanol and air-drying55. Observation is performed on the light microscope 60. Microspodianspores are observable at ×40 based on their darkly stained spore wall,with more characteristics visible at higher magnification.

There are substantial advantages to the present invention. Sensitivityis enhanced. Using protocol # 1, differential coloration of fungi,microsporidia, and Cryptosporidium is obtained, and detected even in thepresence of host cells or other microorganisms, without the need forpurification. Non-chitin containing cells such as bacteria (both Gram−and Gram+) and mammalian cells are selectively decolorized by solution 3in protocol # 1. No purification of chitin-containing organisms isneeded in the present method, as they are apparent and stand out even incomplex mixtures such as septic tank sample, or in presence of bacteriaand mammalian cells. Only a light microscope is needed for observation.

Protocol #2

As shown with reference to FIG. 9, it is within the scope of the presentinvention to modify protocol #1 to establish protocol # 2, in whichsamples are not heated dry before the sequential addition of the dyes atsteps 20 and 25, and observation is made upon addition of a drop ofsolution 3 (SDS) 35 in the middle of the sample. With protocol #2,microsporidia and Cryptosporidium can be identified in the same sample60. The immature microsporidian spores are easily detectable as they areenlarged and elongated while staining in red. C. parvum sporozoites,individual or in bundle, stained by protocol # 2, are dramaticallyenlarged and stained in a distinctive red golden brown. Oocysts arereleased from host cells. Thick walled oocysts do not stain but areapparent, whereas immature oocysts stained in dark red, some of whichare enlarged showing stained merozoites or sporozoites in red goldenbrown. Protocol #2 is allows for the observation of the release ofmicrosporidian spores from parasitophorous vacuoles, followed by agradual migration of these spores to the periphery of the drop of PBSsolution containing SDS, which is accompanied by the germination orelongation of immature spores. Although Cryptosporidium does not containchitin, some staining was observed probably due to the presence ofcomplex carbohydrates such as glucoaminoglycans. Using protocol # 2 tostain mammalian cells infected with C. parvum, intracellular stages, andthin wall-oocysts are released from the infected mammalian cells andstained in a distinctive red golden brown color at the selectivedecoloration step. Hence, microsporidia and Cryptosporidium can berecognized when present in the same sample when stained by protocol # 2.

Protocol #3

In is within the scope of the present invention to modify protocol #1 toestablish protocol # 3 in which other microorganisms, such as bacteria,are differentially stained. In protocol # 3, the selective decolorationstep 35 or 40 is omitted. Apparent differences can be observed betweendifferent strains of E. coli. By omitting the selective decolorationstep, as in protocol # 3, the use of the dyes Ponceau S and Stains-allcan be extended to all other microorganisms for detection based on theirdifference in size, shape and coloration, properties that can bephotographed with a digital camera. For instance, apparent differencesare observed with various Escherichia coli strains. It is noteworthythat a number of modern biosensors are being developed to analyzemicroorganisms based on morphological characteristics.

In summary, the invention is useful for the analysis of fungi, andespecially useful for the detection of microsporidia andCryptosporidium, in pure sample as well as in complex mixture such asclinical and environmental samples, and perhaps also foodstuffs that donot contain chitin. With the invented method, it is possible to detectmicrosporidia and Cryptosporidium in septic tank samples, and samples oforganisms retained on 0.45 um filter from water samples from varioussources or sedimented by centrifugation, as well as infected live orformalin-fixed cells and tissues. Additionally, based on the propertiesmentioned above of the dyes Ponceau S and Stains-all, the presentstaining method can be modified to stain other microorganisms, which canthen be identified based on their size, shape and coloration. Currently,the most specific tests for the identification of microsporidia in watersamples are based on genetic method, for example PCR for theamplification of specific genes (Dowd et al., 1999). In this method, theorganisms have to be isolated, and their DNA extracted. The sensitivityof the method depends on the efficacy of recovery of the spores fromenvironmental samples by immunomagnetic beads, while DNA isolation fromspores is time consuming. This taken with the cost associated with PCRanalysis, it is impractical to use genetic tests to routinely andsystematically monitor water pollution by microsporidia. The stainingmethod developed in the present invention can be used as a presumptivescreening test to identify samples contaminated with microsporidiaand/or C. parvum for further confirmation by genetic analysis. Theentire staining procedure takes less than 30 minutes.

It will be seen that the advantages set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween. Now that theinvention has been described,

What is claimed is:
 1. A kit for detecting the presence of achitin-containing microorganism in a sample suspected of containing themicroorganism, comprising in combination: a) an aqueous solution of 0.1%3-hydroxy-4-[2-sulfo-4-(4-sulfophenylazo)phenylazo]-2,7-naphthalenedisulfonicacid tetrasodium salt in 5% acetic acid in water; b) a solution of1-Ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho[1,2-d]thiazolium bromide in methanol diluted 1:10 in a solution ofacetic acid, methanol and deionized water in a ratio of about 10:40:50,respectively; c) a solution of 0.25% sodium dodecyl sulfate in phosphatebuffered saline for decolorizing; d) a solution of acetic acid,methanol, and deionized water in the ratio of about 10:40:50,respectively for rinsing agent; and e) a solution of phosphate bufferedsaline containing 0.05% Tween 20.